The goal of the proposed research is to develop an optimized microcarrier-bioreactor cell culture system that enables the rapid, efficient, large scale, low cost production of human influenza vaccine. Most human influenza virus vaccine is produced in embryonated eggs. The Federal government and other worldwide organizations are seeking alternative cell-culture approaches to influenza vaccine production since there are inherent difficulties with the egg-derived vaccines. Under normal conditions there are long lead times between (a) the identification of particular virus strains likely in each flu season and (b) the availability of appropriate vaccines against these virus strains. Microcarrier-bioreactor cell-culture technology has the potential to lower production costs and reduce the time required to produce 93 million doses of monovalent vaccine to a few months. This potential to rapidly achieve large scale, would be invaluable in case of an influenza pandemic and possibly could counter a pandemic or bioterrorist acts. To achieve the research goals, five specific aims will optimize microcarrier-bioreactor culture process parameters that affect viral yield with Vero and MDCK cells and develop protocols applicable to economic commercial use. We will (i) license manufacturing protocols to large producers of flu vaccine and (ii) sell Hillex microcarriers for those protocols. [unreadable] [unreadable] Specific Aim I. To identify and integrate critical cell culture and viral growth parameters for optimal production of human influenza virus in Vero cells grown in Hillex microcarrier-suspension culture. [unreadable] Specific Aim II. To extend the range of viral growth parameters (as identified in Specific Aim I) to include six additional strains of influenza virus and to compare the virus production in direct "head-to-head" competition between embryonated eggs and Vero cells grown in Hillex microcarrier-suspension culture. [unreadable] Specific Aim III. To assess production of human influenza virus in Vero cells grown in a microcarrier-bioreactor culture at the one liter scale. [unreadable] Specific Aim IV: To develop a process to expand and scale-up Vero cells from a working seed ampoule to a five liter culture and to demonstrate that the passaged cells will support high influenza virus concentrations at a larger scale. [unreadable] Specific Aim V: To identify and integrate critical cell culture parameters for optimal production of human influenza virus (A/Wuhan/395/95) in MDCK cells grown in Hillex microcarrier-bioreactor culture. [unreadable] [unreadable]